Julia Billiard

The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis

Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display‐polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled‐related protein (sFRP)‐1 was the most highly regulated of these. Transient transfection of HOBs with sFRP‐1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP‐1 mRNA levels increased 24‐fold during HOB differentiation from pre‐osteoblasts to pre‐osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the pre‐osteocytes, which had the highest levels of sFRP‐1 mRNA, also had the highest rate of cell death. Basal sFRP‐1 mRNA levels also increased 29‐fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP‐1 mRNA was induced 38‐fold following prostaglandin E2 (PGE2) treatment of pre‐osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP‐1 expression was down‐regulated by as much as 80% following transforming growth factor (TGF)‐β1 treatment of pre‐osteocytes that had high basal mRNA levels. Consistent with this, treatment of pre‐osteoblasts and mature osteoblasts with PGE2 increased apoptosis threefold, while treatment of pre‐osteocytes with TGF‐β1 decreased cell death by 50%. Likewise, over‐expression of sFRP‐1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP‐1 as an important negative regulator of human osteoblast and osteocyte survival.

Wnt5a induces homodimerization and activation of Ror2 receptor tyrosine kinase

Wnts are secreted glycoproteins that control vital biological processes, including embryogenesis, organogenesis and tumorigenesis. Wnts are classified into several subfamilies depending on the signaling pathways they activate, with the canonical subfamily activating the Wnt/β‐catenin pathway and the non‐canonical subfamily activating a variety of other pathways, including the Wnt/calcium signaling and the small GTPase/c‐Jun NH2‐terminal kinase pathway. Wnts bind to a membrane receptor Frizzled and a co‐receptor, the low‐density lipoprotein receptor related protein. More recently, both canonical and non‐canonical Wnts were shown to bind the Ror2 receptor tyrosine kinase. Ror2 is an orphan receptor that plays crucial roles in skeletal morphogenesis and promotes osteoblast differentiation and bone formation. Here we examine the effects of a canonical Wnt3a and a non‐canonical Wnt5a on the signaling of the Ror2 receptor. We demonstrate that even though both Wnt5a and Wnt3a bound Ror2, only Wnt5a induced Ror2 homo‐dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, Wnt5a treatment also resulted in increased phosphorylation of the Ror2 substrate, 14‐3‐3β scaffold protein, indicating that Wnt5a binding causes activation of the Ror2 signaling cascade. Functionally, Wnt5a recapitulated the Ror2 activation phenotype, enhancing bone formation in the mouse calvarial bone explant cultures and potentiating osteoblastic differentiation of human mesenchymal stem cells. The effect of Wnt5a on osteoblastic differentiation was largely abolished upon Ror2 down‐regulation. Thus we show that Wnt5a activates the classical receptor tyrosine kinase signaling cascade through the Ror2 receptor in cells of osteoblastic origin. J. Cell. Biochem. 105: 497–502, 2008.

A small molecule inhibitor of the Wnt antagonist secreted frizzled-related protein-1 stimulates bone formation

Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone diseases. Deletion of the Wnt antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to sFRP-1 with a K(D) of 0.35 microM in a tryptophan fluorescence quenching assay. This compound also selectively inhibited sFRP-1 with an EC(50) of 3.9 microM in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a K(D) of 0.08 microM and inhibited it with an EC(50) of 0.65 microM. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 microM. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation.

LPA induces osteoblast differentiation through interplay of two receptors: LPA1 and LPA4

The bioactive phospholipid, lysophosphatidic acid (LPA), acting through at least five distinct receptors LPA1–LPA5, plays important roles in numerous biological processes. Here we report that LPA induces osteoblastic differentiation of human mesenchymal stem cells hMSC‐TERT. We find that hMSC‐TERT mostly express two LPA receptors, LPA1 and LPA4, and undergo osteoblastic differentiation in serum‐containing medium. Inhibition of LPA1 with Ki16425 completely abrogates osteogenesis, indicating that this process is mediated by LPA in the serum through activation of LPA1. In contrast to LPA1, down‐regulation of LPA4 expression with shRNA significantly increases osteogenesis, suggesting that this receptor normally exerts negative effects on differentiation. Mechanistically, we find that in hMSC‐TERT, LPA induces a rise in both cAMP and Ca2+. The rise in Ca2+ is completely abolished by Ki16425, whereas LPA‐mediated cAMP increase is not sensitive to Ki16425. To test if LPA signaling pathways controlling osteogenesis in vitro translate into animal physiology, we evaluated the bones of LPA4‐deficient mice. Consistent with the ability of LPA4 to inhibit osteoblastic differentiation of stem cells, LPA4‐deficient mice have increased trabecular bone volume, number, and thickness. J. Cell. Biochem. 109: 794–800, 2010.

Identification of diarylsulfone sulfonamides as secreted frizzled related protein-1 (sFRP-1) inhibitors.

Inhibitor of secreted frizzled related protein-1 (sFRP-1) would be a novel potential osteogenic agent, since loss of sFRP-1 affects osteoblast proliferation, differentiation, and activity, resulting in improved bone mineral density, quality, and strength. We have identified small molecule diarylsulfone sulfonamide derivatives as sFRP-1 inhibitors. Structure-activity relationship generated for various regions of the scaffold was utilized to improve the biochemical profile, resulting in the identification of potent selective analogues, such as 16 with desirable pharmaceutical profile.

Identification of iminooxothiazolidines as secreted frizzled related protein-1 inhibitors.

Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as sFRP-1 inhibitors. Structure-activity relationships were established for various regions of the scaffold along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity.

The role of Wnt signaling in bone and mineral metabolism

Regulation of canonical Wnt signaling in osteoblasts has been shown to play an important role in bone formation. Loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein (LRP)5, cause osteoporosis pseudoglioma syndrome in humans, whereas gain-of-function mutations like G171V lead to high bone mass phenotypes. Mouse models of these conditions have enabled the mechanisms of LRP5 action on bone to be elucidated, and allation of additional pathway components like LRP6, Wnt-10b, and the antagonist secreted frizzled-related protein (sFRP)-1 has extended our understanding of Wnt action in the skeleton. LRP5−/− mice exhibit decreased trabecular bone volume (TBV) at an early age owing to reduced osteoblast proliferation and activity, whereas transgenic LRP5G171V/+ mice demonstrate increased TBV at a young age owing to reduced osteoblast and osteocyte apoptosis. Canonical Wnt signaling also plays a role in mechanosensory stimulation of osteoblasts in vitro, and the LRP5G171V/+ transgenic mice are resistant to disuse-induced bone loss. LRP6−/+ mice display diminished TBV indicating that LRP5 and LRP6 are both required for optimal osteoblast function. Wnt-10b−/− mice also exhibit reduced TBV, demonstrating that this is one of the ligands that controls bone formation. In contrast, sFRP-1−/− mice show heightened TBV, but not until adulthood when enhanced osteoblast proliferation, differentiation and activity, as well as diminished osteoblast and osteocyte apoptosis are observed. sFRP-1 also modulates osteoclast formation in vitro, and other family members like sFRP-4 are able to control phosphate metabolism in vivo. Moreover, anabolic factors like bone morphogenetic protein-2 and parathyroid hormone appear to at least partly control bone formation through intersection with Wnt signaling. Finally, new components of the Wnt pathways like the orphan tyrosine kinase receptor Ror2 have recently been identified as modulators of osteoblast physiology. Thus, Wnt signaling plays a substantial role in the regulation of bone and mineral metabolism. Future research will provide for a better understanding of the mechanisms for Wnt action in the skeleton.

Regulation of Osteoblast Differentiation and Bone Cancers by Wnt and PTH Signaling Pathways

Publisher Summary Osteoblasts arise from mesenchymal stem cells, and the differentiation of these progenitors to osteoblasts is controlled by many growth factors and hormones including Wnts and parathyroid hormone (PTH). The Wnt signaling pathway modulates many cell-fate decisions throughout development by controlling gene expression, cell behavior, adhesion and polarity. Aberrant regulation of the Wnt pathway results in inappropriate expression of target genes that leads to a variety of abnormalities and degenerative diseases and is implicated in several human cancers including osteosarcoma. Studies have indicated that the secreted Wnt antagonist dickkopf-1 inhibits osteoblastic activity, promotes osteoclastic function and plays a role in the pathophysiology of multiple myeloma. Another Wnt antagonist, secreted frizzled-related protein-2, also appears to be involved in this disease. Osteosarcoma is a rare, sporadic malignancy that is found in only 4–5 people per million. These tumors are malignant mesenchymal cancers that produce osteoid and bone, occasionally in a trabecular pattern. Unlike most cancers, osteosarcoma is predominantly a disease of young people that peaks in the second and third decades of life and appears to be associated with the adolescent growth spurt. This cancer is also more common in patients with Pagets disease, and some studies suggest an additional peak after the age of 50. Pre-clinical safety studies with PTH revealed a link between long-term treatment with the peptide and the development of bone neoplasias. The mechanisms for the induction of osteosarcoma by PTH are not well understood but may involve stimulation of osteoblastic cell proliferation and/or prevention of cellular apoptosis.